Cytoprotective effects of diosmetin against hydrogen peroxide-induced L02 cell oxidative damage via activation of the Nrf2-ARE signaling pathway

Abstract

Oxidative stress is considered a crucial mediator in the pathogenesis of various liver diseases. The flavone diosmetin has been reported to exhibit antioxidant activities; however, the hepatoprotective effects of diosmetin against oxidative stress, and the underlying molecular mechanisms, remain unknown. The present study aimed to investigate the potential hepatoprotective effects of diosmetin on hydrogen peroxide (H2O2)-induced oxidative damage in L02 cells and attempted to evaluate the role of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element pathway in this process. L02 cells were divided into groups: Control (DMSO, diosmetin), H2O2, Trolox or tertiary butylhydroquinone and diosmetin (different doses). Protective effects in L02 cells were determined by CCK-8, cell apoptosis and lactate dehydrogenase leakage assays. Flow cytometry and inverted fluorescence microscope were used to measure the intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Protein expression levels were of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) were determined by western blotting and mRNA levels were determined by reverse transcription-quantitative polymerase chain reaction. The results revealed that H2O2 induced notable injury to L02 cells, as demonstrated by decreased cell viability, increased lactate dehydrogenase release, apoptotic rate and intracellular ROS production, and by the loss of MMP. Conversely, diosmetin (20-40 μM) significantly reversed the damaging effects of H2O2, which indicated that diosmetin may exhibit potent hepatoprotective potential against H2O2-induced oxidative damage. Furthermore, pretreatment with diosmetin elevated mRNA and protein expression levels of Nrf2, HO-1 and NQO1. The present study is the first, to the best of our knowledge, to demonstrate that activation of the Nrf2/NQO1-HO-1 signaling pathway maybe involved in the cytoprotective effects of diosmetin against oxidative stress. Therefore, diosmetin may be considered a promising therapeutic agent for the treatment of various liver diseases associated with oxidative stress.